A vast majority of biologically active molecules including growth factors, cytokines, neurotransmitters and hormones transduce signals via specific cell-surface receptors. Some of these receptors are then coupled to heterotrimeric GTP-binding proteins (G proteins) which, upon activation, relay signals to a variety of cellular effectors including at least four phospholipase C (PLC) variants and adenylyl cyclases.
G proteins mediate external signals by forming heterotrimers consisting of an alpha, beta and gamma subunit. Several isoforms of each subunit have been identified and therefore, through subunit heterogeneity, G proteins effectively integrate multiple signaling cascades. The alpha subunits of G proteins contain the GTP binding site and intrinsic catalytic GTPase activity. Based on sequence similarity and function, these subunits have been classified into four major groups; Gs, which stimulate adenylyl cyclases; Gi, which inhibit adenylyl cyclases; Gq, which activate PLC isoforms and G12/13, which mediate pathways associated with cell growth and differentiation (Hamm, J. Biol. Chem., 1998, 273, 669-672).
G-alpha-i2 is a member of the Gi subfamily of G proteins which is involved in hormonal inhibition of adenylyl cyclase and in the regulation of plasma membrane enzymes. It has been shown to activate the Akt/PKB signaling cascade (Murga et al., J. Biol. Chem., 1998, 273, 19080-19085) and to be required in erythropoietin modulation of calcium influx through calcium channels (Miller et al., J. Clin. Invest., 1996, 98, 1728-1736).
Comparison studies of modified forms of the pertussis toxin-insensitive form of G-alpha-i2 (pertussis toxin is normally an inhibitor of Gi function) and the wild type protein in assays designed to investigate the interactions of adrenoceptors and Gi proteins demonstrated that more agonist was required to stimulate the mutant protein than the wild type. These studies showed that the affinity of the wild type Gi protein for the receptor was greater than that of the mutant (Wise et al., Biochem. J., 1997, 321, 721-728).
The expression of G-alpha-i2 has been shown to be altered in some tumors. In human hepatocellular carcinoma (HCC), the expression and functional activity of G-alpha-i2 was increased in 67% of the tumors examined. These results indicate that the regulation of the adenylate cyclase system in these cells may contribute to the formation or progression of the carcinoma (Schmidt et al., Hepatology, 1997, 26, 1189-1194).
Furthermore, unlike other Gi proteins, there exists a mutant form of the G-alpha-i2 protein that may function as an oncogene, GIP2. In this mutation, the Arg179 codon is changed to encode one of several other amino acids. This mutant has been found in tumors of the adrenal cortex and endocrine tumors of the ovary (Lyons et al., Science, 1990, 249, 655-659).
G-alpha-i2 may also play a role in the development of chemical dependence as the expression of the mRNA was increased in the hippocampus after repeated methamphetamine administration in rats (Ujike et al., Neuroreport., 1996, 7, 2036-2040).
Finally, the expression of G-alpha-i2 has been shown to be regulated during physiological shear stresses such as flow. Using a transcapillary coculture system, it was shown that G-alpha-i2 expression was decreased by high-flow conditions in endothelial and vascular smooth muscle cells implicating Gi proteins in flow-induced responses of vessel wall function (Redmond et al., Arterioscler. Thromb. Vasc. Biol., 1998, 18, 75-83).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of G-alpha-i2 and strategies aimed at inhibiting or investigating G-alpha-i2 function have involved the use of several techniques.
Mice lacking the G-alpha-i2 gene display growth retardation and develop adenocarcinoma of the colon and a form of lethal diffuse colitis similar to ulcerative colitis in humans (Rudolph et al., Nat. Genet., 1995, 10, 143-150).
Antibodies to G-alpha-i2 were used to measure the reduction of protein in brown adipose tissue when sensitized with noradrenaline. The results showed a 50% decrease in protein implicating the Gi protein in the desensitation process (Svoboda et al., Biochem. J., 1996, 314, 761-768).
In addition, an upstream inhibitor of phospholipase C, U73122, resulted in the inhibition of Gi-mediated protein activation (Wu et al., Neuroreport., 1998, 9, 99-103).
Antisense vectors targeted to G-alpha-i2 have been used to illustrate the role of G-alpha-i2 in cell differentiation (Watkins et al., Science, 1992, 258, 1373-1375), adipogenesis (Wang and Malbon, Int. J. Obes. Relat. Metab. Disord., 1996, 20 Suppl 3, S26-31), and neonatal growth (Moxham et al., Science, 1993, 260, 991-995).
These strategies, however, are untested as therapeutic protocols. Consequently there remains a long felt need for additional agents capable of effectively inhibiting G-alpha-i2 function and the administration of antisense oligonucleotides may provide a promising new pharmaceutical tool for the effective and specific modulation of G-alpha-i2 expression.